The front-line of clinical immunotherapeutic regimens includes patient immunizations against infectious pathogens and other health-threatening agents. Despite the plethora of immunization agents, inoculations may afford, at best, partial immunity, requiring frequent re-immunizations. Such is the case for various conventional monovalent or polyvalent vaccines. And even among such vaccines, the number of single agent inoculants capable of eliciting immunity against a variety of immunogens is limited. Furthermore, antigenic variation among pathogens may limit the efficacy of conventional vaccines.
Due to such obstacles, efforts have focused on methodologies for enhancing the immune system response to given immunogens. To that end, immunogenic conjugates have been produced by linking immunogens to hepatitis B virus (“HBV”) core particles (also referred to as nucleocapsids or nucleocapsid shells), in efforts to enhance the immunogenicity of the linked immunogen, through the operation of T cell dependent and T cell independent determinants of HBV core antigen. See, for example, U.S. Pat. No. 4,818,527 and R. Ulrich et al., “Core Particles of Hepatitis B Virus as Carrier for Foreign Epitopes”, Adv. Virus. Res., 50, pp. 141-82 (1998). Enhanced immunogenicity of epitopes of interest has also been approached via hybrid viral particle-forming proteins, comprising at least a portion of a naturally occurring viral particle forming protein, for example HBV surface antigen, and one or more epitopic sites of interest. See U.S. Pat. No. 5,965,140. As evident from such efforts, proteins of HBV have been used as platforms for presenting immunogens of interest to the immune system.
Hepatitis B virus is a blood-borne virus, comprising a small, partially double-stranded DNA genome, carrying four extensively overlapping open reading frames, consisting of an inner nucleocapsid, comprising the HBV core protein (“HBcAg”), viral polymerase and viral DNA, surrounded by a membranous envelope containing HBV surface antigens (“HBsAg”). The viral envelope contains three different, but related surface antigen proteins, long (L), medium (M) and short (S), which share a common carboxy terminal region but have different amino termini, arising from variable use of initiation triplets at different points within a continuous open reading frame.
The long polypeptide (L polypeptide) consists of pre-S1, pre-S2 and S regions. It is the product of the entire reading frame and comprises the pre-S1 domain of 108 amino acids (or 199, depending on the virus subtype) at its amino terminus, followed by the pre-S2 domain of 55 amino acids, and the short polypeptide (S polypeptide) region of 226 amino acids. The medium-length polypeptide (M polypeptide) has the pre-S2 domain at its amino terminus followed by the S region, whereas the S polypeptide, which is the most abundant form, consists of only the S region. The pre-S regions are believed to play an important role in both viral assembly and attachment to the host cell. The S form is more abundant than the M and L forms of HBsAg in the virus, and occurs in both glycosylated and nonglycosylated forms [V. Bruss and D. Ganem, “The Role of Envelope Protein in Hepatitis B Virus Assembly”, Proc. Natl. Acad. Sci. USA, 88, pp. 1059-63 (1991); V. Bruss et al., “Post-translational Alteration in Transmembrane Topology of Hepatitis B Virus Large Envelope Protein”, EMBO J., 13, pp. 2273-79 (1994); A. R. Neurath et al., “Identification and Chemical Synthesis of a Host Cell Receptor Binding Site on Hepatitis B Virus”, Cell, 46, pp. 429-36 (1986); K. Ueda et al., “Three Envelope Proteins of Hepatitis B Virus: Large S, Middle S and Major S Proteins Needed for the Formation of Dane Particles”, J. Virol., 65, pp. 3521-29 (1991)]. Specific interactions between the outer surface of the core and the inner surface of the envelope are likely to guide correct assembly of the virus and stabilize the resulting particle
HBV core protein can be expressed efficiently in E. coli [M. Pasek et al., “Hepatitis B Virus Genes and Their Expression in E. coli”, Nature, 282, pp. 575-79 (1979)], where it assembles into icosahedral shells of two sizes containing either 180 (T=3) or 240 (T=4) subunits [R. A. Crowther et al., “Three-Dimensional Structure of Hepatitis B Virus Core Particles Determined by Electron Microscopy”, Cell, 77, pp. 943-50 (1994)]. The subunits are clustered as dimers and each dimer forms a spike which protrudes on the surface of the shell. Using electron cryomicroscopy and image processing, a map of the T=4 shell was recently made at 7.4 Å resolution from images of more than 6000 individual particles [B. Böttcher et al., “Determination of the Fold of the Core Protein of Hepatitis B Virus by Electron Cryomicroscopy”, Nature, 386, pp. 88-91 (1997)]. This revealed the fold of the polypeptide chain, which was largely α-helical and quite unlike previously solved viral capsids. Each dimer spike was formed by a pair of long α-helical hairpins, one from each monomer in the dimer [Böttcher et al. (1997); J. F. Conway et al., “Visualization of a 4-Helix Bundle in the Hepatitis B Virus Capsid by Cryo-electron Microscopy”, Nature, 386, pp. 91-94 (1997)]. A numbering scheme which superimposed the amino acid sequence on the fold [Böttcher et al. (1997)] placed the major immunodominant region of the HBV core protein around amino acids 78-82 [J. Salfeld et al., “Antigenic Determinants and Functional Domains in Core Antigen and E Antigen from Hepatitis B Virus”, J. Virol, 63, pp. 798-808 (1989); M. Sällberg et al., “Characterisation of a Linear Binding Site for a Monoclonal Antibody to Hepatitis B Core Antigen”, J. Med. Virol., 33, pp. 248-52 (1991)], at the tip of the spike.
Agents which inhibit HBV viral assembly include those that bind to the core antigen of HBV, thereby blocking the interaction between HBV core proteins and HBV surface proteins. Some such HBV capsid-binding peptides are described in PCT patent application WO98/18818 and in M. R. Dyson and K. Murray, “Selection of Peptide Inhibitors of Interactions Involved in Complex Protein Assemblies: Association of the Core and Surface Antigens of Hepatitis B Virus”, Proc. Natl. Acad. Sci. USA, 92, pp. 2194-98 (1995).
As will be apparent from the disclosure to follow, HBV capsid-binding peptides may be advantageously used as ligands for constructing HBV core antigen particles characterized by the ability to elicit enhanced immune responses to single or multiple immunogens.